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PCV2 (porcine circovirus 2) ELISA (enzyme-linked immuno sorbent assay) antigen detection kit as well as preparation method and applications thereof

A porcine circovirus and antigen detection technology, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problem that the specificity or sensitivity of the kit cannot meet the market detection and other problems

Active Publication Date: 2013-04-10
WUHAN CHOPPER BIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The kit disclosed in this document has not been marketed so far, and the reason for the analysis may be that the specificity or sensitivity of the kit cannot meet the needs of market detection

Method used

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  • PCV2 (porcine circovirus 2) ELISA (enzyme-linked immuno sorbent assay) antigen detection kit as well as preparation method and applications thereof
  • PCV2 (porcine circovirus 2) ELISA (enzyme-linked immuno sorbent assay) antigen detection kit as well as preparation method and applications thereof
  • PCV2 (porcine circovirus 2) ELISA (enzyme-linked immuno sorbent assay) antigen detection kit as well as preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1 Construction of recombinant baculovirus

[0084] The Bac-to-Bac system was used to construct a recombinant baculovirus, and the following primers were designed according to the gene sequence published by GENBANK (NCBI accession number EU340257.1):

[0085] P1: TCTGGATCCATGACGTATCCAAGGAGGCG

[0086] P2: GCGAAGCTTTAAGGGTTAAGTGGGGGGTC

[0087] P3: TCTCTCGAGATGACGTATCCAAGGAGGCG

[0088] P4: GCGGGTACCTAAGGGTTAAGTGGGGGGTC

[0089] Using the PCV2b strain ORF2 sequence as a template (the template was synthesized according to the published PCV2b ORF2 sequence: accession number EU340257.1), P1, P2 amplify the PCV2ORF2 gene, and clone the gene into the pMD-19T vector to obtain a recombinant The vector pMD-19T-ORF2-1 uses the PCV2b strain ORF2 sequence as a template, P3, P4 amplify the PCV2ORF2 gene, and clone the gene into the pMD-19T vector to obtain the recombinant vector pMD-19T-ORF2-2.

[0090] PMD-19T-ORF2-1 was digested with BamH I and Hind III, the ORF2 gene was cloned ...

Embodiment 2

[0091] Example 2 Serum-free suspension culture of insect cells in a bioreactor and quantitative expression of Cap protein

[0092] Culture Sf9 insect cells aseptically in a 1000ml shake flask for 3-4 days, until the concentration grows to 3-5×10 6 cells / ml, when the viability is greater than 95%, inoculate the cells into a 5L bioreactor with a concentration of 3-8×10 5 cells / ml. When the cell concentration reaches 3-5×10 6 When cells / ml, inoculate the cells into a 50L bioreactor and wait until the cells grow to a concentration of 3-5×10 6 cells / ml, inoculate into a 500L bioreactor, until the cell concentration reaches 2-8×10 6 In cells / ml, inoculate recombinant virus rBac-2ORF2 or rBac-ORF2, the multiplicity of infection (MOI) is 0.001-10, the reactor culture condition is pH 6.0-6.5, temperature 25-27℃, dissolved oxygen 30-80%, stirring Speed ​​100-180rpm. Considering the optimal conditions for cell culture, the preferred pH is 6.2, the cell culture stage temperature is set ...

Embodiment 3

[0098] Example 3 Purification of VLP particles and observation by electron microscope

[0099] Harvest the expression cell culture, centrifuge the cell culture at 10000g at 4°C for 30min, remove the cell debris, take the supernatant, centrifuge the supernatant at 31000rpm for 3h (Beckman SW70 rotor), resuspend the pellet with a small amount of PBS, and wait until the pellet is completely dissolved , Add CsCl according to 2.1g / 4.5ml solution, mix evenly, dispense into 5ml ultracentrifuge tube, add PBS, to the distance 2-3mm from the tube mouth, after accurate balance, 149000g, 10℃ centrifugal 24h. After centrifugation, two light yellow zona pellucida can be seen. The target zone (lower zone) is sucked out and collected, which is the purified virus-like particle, which is the PCV2-Cap protein. See the test results Image 6 .

[0100] Take the same amount of cell cultures expressing rBac-ORF2 and rBac-2ORF2 under the same conditions, and process them as described above, dilute t...

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Abstract

The invention relates to a PCV2 (porcine circovirus 2) ELISA (enzyme-linked immuno sorbent assay) antigen detection kit as well as a preparation method and applications thereof, wherein the detection kit comprises an elisa plate of a polyclonal antibody of peridium anti-PCV2-Cap (nucleocapsid) protein, seal liquids, sample diluent, an antigen standard product, a second antibody of a monoclonal antibody of HRP marked anti-PCV2-Cap protein, a concentrated washing liquid, an enzyme substrate solution A, an enzyme substrate solution B and a stop solution, wherein the antigen standard product is purified reconstructed PCV2-Cap protein. The specificity of the kit provided by the invention achieves 100%, and the sensitivity is as high as 4ng / ml, and the kit can be used for swinery PCV2 antigen detection and PCV2 vaccine product quantitative detection.

Description

Technical field [0001] The invention relates to a detection kit, in particular to a porcine circovirus type 2 ELISA antigen detection kit and a preparation method and application thereof. Background technique [0002] Porcine circovirus type 2 (PCV2) is the main pathogen causing postweaning multisystemic wasting syndrome (PMWS) in weaned piglets. In addition to causing PMWS, PCV2 is also associated with piglet congenital tremor, porcine dermatitis nephritis syndrome, porcine respiratory syndrome (PDNS), sow reproductive disorders and other diseases. Since PMWS was first discovered in Canada in 1996, related diseases caused by PCV2 infection have been widespread all over the world. Up to now, the harm of PCV2 infection in my country's pig industry has become increasingly prominent. Therefore, the clinical need to develop PCV2 antigen detection kits is increasingly urgent. [0003] At present, the PCV2 detection methods used at home and abroad mainly include virus isolation and cu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577C12N15/866C12R1/93
Inventor 廖园园漆世华朱薇李建秦伟王威熊媛媛张萍秦红刚李伟谢红玲温文生王桢桢靖志强吴玉石韩兴刘洁
Owner WUHAN CHOPPER BIOLOGY
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